As recently reviewed by us, SERT-deficient +/- and -/- mice have gene-proportionate increases in extracellular fluid serotonin concentrations, i.e., 3- or 6-fold excesses respectively over wildtype +/+ mice. At the same time, SERT -/- mice have a marked deficit of intracellular, releasable serotonin. Despite increased extracellular levels of serotonin and decreased serotonin clearance, SERT +/- mice have unchanged tissue serotonin concentrations in the brain and periphery, and unchanged brain serotonin synthesis and turnover. Thus, a single copy of Slc6a4 is adequate to maintain overall serotonin tissue homeostasis. Continuing advances have been made in our studies of serotonin-related toxic reactions, including the serotonin syndrome. Most commonly, this toxicity occurs as a side effect in humans treated with certain antidepressant and anti-anxiety drugs. Importantly, its milder forms contribute to reduced therapeutic efficacy or a requirement to interrupt treatment in some individuals treated with SRIs. Our earlier studies exploring this behavioral and temperature-related syndrome in SERT-deficient mice revealed a genetic vulnerability to a markedly exaggerated serotonin syndrome when these mice were exposed to the metabolic precursor of serotonin, 5-HTP, or to other serotonergic drugs such as the monoamine oxidase-inhibiting (MAO-I) antidepressant tranylcypromine. Tranylcypromine is a clinically available antidepressant drug, and 5-HTP is readily available on internet sites and sold over-the-counter as a dietary supplement, and thus can be misused. We furthered these initial studies to show that SERT-deficient mice also have exaggerated behavioral responses to the atypical opioids tramadol and meperidine, prescribed as anti-pain medications. This past year, we have expanded our serotonin syndrome studies to investigate recently acquired MAO-AB knockout mice, and demonstrated exaggerated responses to 5-HTP and tramadol in these mice. In addition to the serotonin syndrome behavioral changes, exaggerated alterations in temperature responses were also found in SERT- and MAO-deficient mice. We are now further extending these studies to include dopamine transporter (DAT) knockout mice. Multiple serotonin receptors were discovered to be involved in these exaggerated behavioral and temperature responses. Specifically, in SERT-deficient mice, the exaggerated behavioral responses were mediated by postsynaptic 5-HT1A receptors. Further, whereas in SERT +/+ and +/- mice the 5-HT1A autoreceptor was primarily involved in temperature responses to 5-HTP, in SERT -/- mice the 5-HT7 receptor was the primary mediator of temperature changes, with interesting 5-HT7/5-HT1A interactions revealed. As roles for 5-HT7 receptors in anxiety and depression were recently established, and contributions of 5-HT1A and other serotonin receptors to therapeutic responses to serotonergic drugs have been well-documented, the current findings are relevant to the treatment of human anxiety and affective disorders. These findings in these mouse genetic models suggest the likelihood that humans with lower-expressing SLC6A4 SS genotypes, or other SERT or MAO variants that may lead to 50-80% decreases in SERT binding sites or transport function, may be at higher risk to develop serotonin syndrome neurotoxicity. This is based on highly congruent data from imaging, neuroendocrine, and other studies that have compared SERT-deficient mice, and less commonly SERT-deficient non-human primates, to humans with SERT and MAO gene variants. Of special note, it is likely that relatively mild serotonin syndrome occurrence may contribute to early discontinuation of SRIs and other side effects during SRI treatment that are strongly associated with the lower-expressing SLC6A4 SS/LgLg genotypes and S or Lg alleles, as well as other newly discovered lower-expressing variants in this gene, as reviewed this year and as described in our other report, MH000336-32 LCS. Given this transgenic mouse data and human SLC6A4 and MAO polymorphism data, we have formed an international collaborative effort - "Genes Involved in SEroTonin Toxicity" or "GISETTO" - in which we are examining functional variants in the multiple serotonin (SLC6A4, MAOA) and drug metabolizing gene groups (e.g., CYP2D6) in individuals developing serotonin toxicity and other serotonin-related toxicities, both retrospectively and prospectively. Understanding these genetic mechanisms will improve serotonergic drug safety and efficacy by identifying patients whose response to drugs might be genetically compromised, and thereby might be at risk of adverse and potentially life threatening drug reactions. SERT and MAO provide intriguing examples of likely mouse-human congruence in genetic vulnerability to serotonin toxicity features. Functional variants exist in additional genes that can also be postulated to confer vulnerability to serotonin neurotoxicity, for example, serotonin receptor genes. In addition to our examinations of the serotonin syndrome and related receptor mediation, we have discovered several other alterations in serotonin receptors in SERT-deficient mice. For example, SERT-deficient mice show abnormalities in serotonin 5-HT2C receptors that are expressed in amygdala, a brain region involved in anxiety and fear responses. Specifically in the three-chamber social interaction test, the 5-HT2C agonist mCPP decreased sociability and sniffing in SERT +/+ mice, indicative of the well-documented anxiogenic effect of this drug. This response was absent in SERT -/- mice. Likewise, in the open field test, the selective 5-HT2C agonist RO 60-0175 induced an anxiogenic response in SERT +/+ mice, but not in SERT -/- mice. Since 5-HT2C receptor pre-mRNA is adenosine-to-inosine edited, we evaluated the 5-HT2C receptor RNA editing profiles of SERT +/+ and SERT -/- mice in amygdala. Compared to SERT +/+ mice, SERT -/- mice showed a decrease in less edited, highly functional 5-HT2C isoforms, and an increase in more edited isoforms with reduced signaling efficiency. This increased RNA editing could explain, at least in part, the decreased behavioral responses to 5-HT2C agonists in SERT -/- mice. These alterations in 5-HT2C receptor in amygdala may be relevant to humans with SERT and/or 5-HT2C polymorphisms. In addition to alterations in 5-HT2C receptors, we also recently reported further changes in 5-HT2A receptors in SERT-deficient mice, as these mice show reduced head twitch responses to serotonergic agents such as the 5-HT2A receptor agonists and hallucinogen DOI and the new 5-HT2A agonist TCB-2. The head twitch response in rodents is considered a pharmacologic proxy for hallucinogenic effects, as most of the drugs that induce this response in rodents are hallucinogens in humans, and these responses can be blocked by anti-psychotic drugs. In exploring mechanisms underlying this response, we found that reducing excess brain serotonin in SERT-deficient mice by using a serotonin synthesis inhibitor restored the deficient head twitch state in SERT -/- mice to levels of control SERT +/+ mice. These findings provide a more comprehensive understanding of mechanisms in this genetic mouse model that eventually may provide insights into human disorders with genetic contributions that include hallucinogenic phenomena such as schizophrenia and bipolar disorder, as well as the consequences of certain types of substance abuse disorders - all of high impact, and major public health concerns. Overall, the data accumulated by our Lab, as referenced below and previously, support the use of different genetically modified mice as vulnerability models for humans with SERT, MAO and other gene variants with regard to gene-gene and gene-environment interactions that contribute to human diseases and their pharmacologic treatment.